PROJECT SUMMARY! More than 1.4 million people are diagnosed with colorectal cancer (CRC) each year, and while increased rates of endoscopic screening have led to a slight reduction in mortality, the disease still accounts for more than 600,000 deaths worldwide annually. Compounding this problem, in recent years there has been a disproportionate increase in early onset CRC, highlighting it as a major, ongoing public health problem. Treatments for CRC have changed little over the past 10-15 years, and we still have no effective targeted therapies for the majority of CRC patients; new strategies are urgently needed. Hyperactivation of the WNT signaling pathway is a hallmark and major oncogenic driver of CRC, occurring in ~95% of tumors. Indeed, there is strong evidence from pre-clinical model systems that targeting hyperactive WNT signaling can provide significant therapeutic benefit in CRC. However, unlike many other oncogenic signaling networks, controlling WNT pathway activity with drugs has proven quite challenging. Relatively recent work has drawn attention to Tankyrase (TNKS) enzymes as an exciting therapeutic target in CRC. These enzymes positively regulate WNT signaling levels, but their activity can be inhibited by certain small molecule inhibitors. Early work has demonstrated that TNKS inhibitors can suppress hyperactive WNT signaling and impede cancer cell proliferation; however, those same inhibitors are extremely toxic in vivo, due to their suppression of WNT signaling in normal intestinal stem cells. Tumors often contain significant disruptions to their genomes, including gains and losses of large chromosome segments. These losses are presumed to support cancer cell growth, but also lead to loss of `passenger' genes that do not drive cancer progression, but may unintentionally `rewire' the signaling networks. I will test the hypothesis that large chromosomal deletions, common in CRC, create a tumor-specific vulnerability for selective inhibition of one specific Tankyrase family member ? TNKS2. Because normal cells do not carry these tumor-associated deletions, they should remain unaffected when inhibiting this single TNKS enzyme. In Aim 1, using a combination of new CRISPR-based genome editing technology, with inducible and reversible shRNA tools, I will determine how heterozygous and homozygous chromosome deletions impact the response to TNKS2 inhibition. Further, I aim to test small molecule inhibitors that selectively target TNKS2. In Aim 2 I will utilize a unique transgenic shRNA technology, developed by my mentor, to examine the toxicity and efficacy of selective TNKS2 inhibition in vivo. This genetic approach is powerful, as it allows an assessment of potent and systemic gene silencing without the limitation of poor drug delivery or specificity. Identifying a safe and effective approach to disrupt hyperactivated WNT signaling will have a profound impact on the clinical management of CRC. Thus, we believe our work will contribute significant pre-clinical data to develop safe and effective targeted therapies for CRC.